DESCRIPTION: Cystic fibrosis (CF) gene therapy can potentially benefit all CF patients, regardless of the specific gene mutation leading to the disease state. However, gene therapy trials, using viral and non-viral vectors, have had disappointing outcomes to date due to inability to overcome biological barriers, including the purulent sputum gel layer, periciliary layer and epithelial surface. In particular, we previously found that several clinically and preclinically teted viral and non-viral gene vectors are trapped in human CF sputum, which prevents them from reaching and delivering their therapeutic gene cargo to the underlying airway epithelium. We recently developed two delivery strategies: (i) ultra-small DNA-loaded nanoparticles, called mucus-penetrating DNA nanoparticles (DNA-MPP) that efficiently penetrate human CF sputum and (ii) mildly hypotonic aqueous vehicles, which further improves DNA-MPP penetration through the sputum gel layer and periciliary layer via osmosis-driven fluid absorption, and enhances DNA-MPP uptake by the airway epithelium via the regulatory volume decrease mechanism. We found that the combination of these two approaches led to efficient gene transfer to the airway epithelium of normal mice in vivo and primary human airway cells in vitro. Here we propose to further develop and validate our combined approach in relevant in vitro and in vivo settings. To this end, we will evaluate CFTR gene transfer to CF human primary airway epithelium grown at air-liquid interface and in the lung airways of CF mouse model thoroughly characterized for its CF-like lung diseases. We will also determine whether our delivery strategies allow persistent transgene expression upon a single and repeated dosing without eliciting acute and chronic toxicity. If we are successful, we will test our strategies in macaques and potentially clinical studies in the future.